Subproject 1We have previously identified novel protease inhibitors (PIs) containing 3(R),3a(S),6a(R)-bis-tetrahydrofuranyl urethane (bis-THF) in collaboration with Professor Arun Ghosh of Purdue University, West Lafayette, Indiana (Yoshimura & Mitsuya et al. J Virology 76:1349-1358, 2002; Ghosh & Mitsuya et al. J Med Chem 49:5252-5261, 2006). One of such bis-THF-containing PIs (Koh & Mitsuya et al. Antimicrob Agents Chemother. 47:3123-3129, 2003) is called TMC114, which was found to be superior to other protease inhibitors in highly drug-experienced individuals in recently completed clinical trials. TMC114 was approved by the US Food and Drug Administration (FDA) under a generic name darunavir (PREZISTA) as a prescription drug for those who harbor multiple drug resistant HIV variants and do not respond to any previously existing combination regimens. The P.I. of the Section is an inventor in this discovery effort of darunavir with others in the NCI's patent application (DHHS Reference E-200-98/3; US Patent Application No. 09/720,276). In the report period, we have also explored multiple scaffolds for the design of PIs, generating cycloamide-derived PIs (Ghosh & Mitsuya et al. J Med Chem 48:3576-3585, 2005), PIs incorporating oxyindoles (Ghosh & Mitsuya et al. Bioorg. Med. Chem. Lett. 16:1869-1873 2006), and other bis-tetrahydrofuran-containing PIs (Ghosh & Mitsuya et al. Chem Med Chem 1:939-950, 2006). Continuing further collaboration with Dr. Ghosh and his group, we are developing more effective and resistant HIV-"repellant" PIs containing unique active components.In another area of drug development, we have also identified a number of novel nucleoside analogs, including 2',3'-dideoxy-congeners of 4'-ethynyl-nucleoside derivatives (Ohrui & Mitsuya et al. J Med Chem. 43:4516-4525, 2000). One of such analogs, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), has proved to be highly potent against multi-nucleoside reverse transcriptase inhibitor-resistant HIV-1 variants. In particular, we have demonstrated EFdA, once triphosphorylated in the cytoplasm of target cells, remains within cells, persistently exerting potent antiviral activity against the virus, strongly suggesting that once or twice daily regimens are possible with EFdA. We are presently forwarding to pre-clinical testing, in which EFdA is intraperitoneally administered twice a day to human PBM-transplanted SCID mice.Subproject 2CCR5 is one of the two co-receptors besides CD4 molecules, which HIV-1 exploits in its entry to target cells, thereby serving as attractive targets for possible intervention of HIV infection. It is of note that considering that if successfully developed, CCR5 inhibitors could "resist" to the emergence of resistant virus, because the target for CCR5 inhibitors is indeed a cellular apparatus, therefore, HIV-1 cannot use the same strategy to acquire resistance to RTIs and PIs: "to change itself". We thus redirected our attentions from the NRTI- and PI-related research lines and started our efforts to identify such entry inhibitors. In collaboration with scientists of Ono Pharmaceutical Co., Ltd., which is based in Osaka, Japan, we identified a new class of CCR5 inhibitors, spirodiketopiperazine (SDP) derivatives. SDP derivatives bind to CCR5, block CC-chemokine(CC)/CCR5 interactions, and inhibit HIV-1 infection of CCR5+ cells (Maeda & Mitsuya et al. J Biol Chem. 276:35194, 2001). One such CCR5 inhibitor aplaviroc (AVC) exerted highly potent activity against a wide spectrum of laboratory and primary R5-HIV-1 isolates including multi-drug resistant HIV vaiants (IC50 values of 0.2-0.6 nM), which is associated with its high CCR5-binding affinity (KD values of 2 nM) and potent inhibition of CCR5-gp120 binding. AVC, once bound to CCR5+ human cells, remained on the cell surface for >9 hr after thorough washing, and blocked R5-HIV infection upon delayed HIV-1 exposure.